Wayne B. Borth

The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with unique activities.

The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses (Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS.

Characterization of a Virus Infecting Citrus volkameriana with Citrus Leprosis-Like Symptoms

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco

Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1Xoo and Hpa1Xoc, two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1Xoo and Hpa1Xoc, respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H2N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal α-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1Xoo (L51P) and Hpa1Xoc (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.

An assemblage of closteroviruses infects Hawaiian ti (Cordyline fruticosa L.)

The ti plant (Cordyline fruticosa L.) is culturally important throughout most of Polynesia and has considerable economic importance in Hawai’i where the foliage is commonly used in cultural ceremonies as well as food and ornamental industries. In Hawai’i, ringspot symptoms were recently observed on leaves of the common green variety of ti growing in Kahalu’u on the island of O’ahu, and Wailuku and Hana on the island of Maui. High molecular weight double-stranded (ds)RNAs were isolated from the leaves of symptomatic plants as well as plants without symptoms. A cDNA library derived from the dsRNAs present in symptomatic plants was generated and sequenced. These sequences indicated at least four distinct clostero-like viruses were present in the plants, and phylogenetic analyses suggested they were most closely related to Little cherry virus 1, an unassigned member of the family Closteroviridae. The 16,883 nucleotide genome of one of these viruses was determined and predicted to contain ten open reading frames with an organization typical of closteroviruses. Reverse-transcription PCR revealed this virus was present in both symptomatic and asymptomatic ti plants, making it unlikely to be responsible for the observed ringspot symptoms. We propose the name Cordyline virus 1 (CoV-1) for this virus and include it as a new, unassigned member of the family Closteroviridae.

Molecular characterization of closteroviruses infecting Cordyline fruticosa L. in Hawaii

In Hawaii, common green ti plants (Cordyline fruticosa L.) have been shown to harbor Cordyline virus 1 (CoV-1) which, along with Little cherry virus 1 (LChV-1), and Grapevine leafroll-associated virus 7 (GLRaV-7), form a distinct clade within the family Closteroviridae. Preliminary work has indicated that, aside from CoV-1, three additional closteroviruses may infect common green ti plants in Hawaii. In this study, pyrosequencing was used to characterize the genomes of closteroviruses infecting a single common green ti plant. The sequence data confirmed the presence of CoV-1 as well as three additional closteroviruses. Although all four viruses had the same general genome organization, the sequence divergence between the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein ranged from 22 to 61%, indicating these represent four distinct closterovirus species. The names CoV-2, CoV-3, and CoV-4 are proposed for the three new viruses. Phylogenetic analyses placed CoV-2, CoV-3, and CoV-4 in the same clade as CoV-1, LChV-1, and GLRaV-7.

Effects of Synthetic Cecropin Analogs on in Vitro Growth of Acholeplasma laidlawii

ABSTRACT Four synthetic peptides (Peptidyl MIMs; Demeter Biotechnologies, Inc.) were evaluated for their in vitro activity againstAcholeplasma laidlawii. Fifty percent effective concentration values ranged from 1 to 15 μM. Three of these compounds are more lethal than cecropin B against A. laidlawii.

Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

Differentiation and Distribution of Cordyline Viruses 1-4 in Hawaiian ti Plants (Cordyline fruticosa L.)

Common green ti plants (Cordyline fruticosa L.) in Hawaii can be infected by four recently characterized closteroviruses that are tentative members of the proposed genus Velarivirus. In this study, a reverse-transcription polymerase chain reaction (RT-PCR) assay developed to detect and distinguish Cordyline virus 1 (CoV-1), CoV-2, CoV-3, and CoV-4 was used to determine: (i) the distribution of these viruses in Hawaii; and (ii) if they are involved in the etiology of ti ringspot disease. One hundred and thirty-seven common green ti plants with and without ti ringspot symptoms were sampled from 43 sites on five of the Hawaiian Islands and underwent the RT-PCR assay. Eleven ornamental ti varieties were also sampled and assayed. Based on this survey, it appears none of the CoVs are involved in the etiology of ti ringspot. The observation of a non-uniform geographic distribution of the CoVs in common green ti, combined with the presence of CoVs in seed-derived ornamental varieties, suggests active vector transmission. Eight herbarium specimens collected between 1903 and 2003 from plants on the island of Oahu also underwent the RT-PCR assay. Amplifiable RNA was isolated from accessions collected in 1985 or later, however only the 2003 accession was found to harbor CoVs.

Molecular Characterization and Distribution of Two Strains of Dasheen mosaic virus on Taro in Hawaii

Dasheen mosaic virus (DsMV) is one of the major viruses affecting taro (Colocasia esculenta) production worldwide. Whole genome sequences were determined for two DsMV strains, Hawaii Strain I (KY242358) and Hawaii Strain II (KY242359), from taro in Hawaii. They represent the first full-length coding sequences of DsMV reported from the United States. Hawaii Strains I and II were 77 and 85% identical, respectively, with other completely sequenced DsMV isolates. Hawaii Strain I was most closely related to vanilla mosaic virus (VanMV) (KX505964.1), a strain of DsMV infecting vanilla in the southern Pacific Islands. Hawaii Strain II was most closely related to a taro DsMV isolate CTCRI-II-14 (KT026108.1) from India. Phylogenetic analysis of all available DsMV isolates based on amino acid sequences of their coat protein showed some correlation between host plant and genetic diversity. Analyses of DsMV genome sequences detected three recombinants from China and India among the six isolates with known complete genome sequences. The DsMV strain NC003537.1 from China is a recombinant of KJ786965.1 from India and Hawaii Strain II. Another DsMV strain KT026108.1 is a recombinant of Hawaii Strain II and NC003537.1 from China. The third DsMV strain KJ786965.1 from India is a recombinant of Hawaii Strain II and NC003537.1 from China. To our knowledge, this is the first report of recombination events in DsMV. Both Hawaii Strains I and II of DsMV were found widespread throughout the Hawaiian islands.

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.